Agrobacterium Mediated Transformation Of Soybean System Optimization And Report Gene DsRed2Expression Efficiency Evaluation

Soybean is an important oil crops and cash crops, is the chief crop in edible oil andprotein source, played important roles in the food industry and agriculture production.With the continuous development of genetic engineering technology, the use ofgenetically modified soybean varieties, new germplasm has been widely used. In ourcountry, the research on genetically modified crops are still in the initial stage, therefore,strengthening independent research and development of genetically modified soybeansin our country, improve the efficiency of soybean genetic transformation is the key ofthe soybean genetic improvement in our country.In order to improve the soybean plant regeneration system, improve the efficiency ofagrobacterium mediated soybean cotyledon section system transformation, establish astable and efficient transformation system, the experiment of different genotypes andgermination time under the condition of multiple shoot clumps of inducing rate,elongation, and the instantaneous rate of statistics, select the optimal genotype andgermination time.This study chose Jack, William82, Zhonghuang13, Zigongdongdou and Jilin no.1offive strains, in the different germination time under the induction of regeneration wereanalyzed. Analysis results show that Jack strain section as soybean cotyledon explant,the regeneration efficiency is high and stable growth, the most suitable for conversionexperiment. And then on germination of3to8days Jack strains of soybean cotyledonas explant, the plantlet regeneration and agrobacterium infect root carcinomaexperiment, statistics the regeneration process, multiple shoot clumps of inducing rate,elongation and instantaneous positive rate. Experimental results show that thegermination of soybean cotyledon do3day for as explant regeneration system andtransformation system, the multiple shoot clumps of inducing rate, elongation andinstantaneous positive rate were higher than other days, show significant difference.Today, the plant gene transformation system is the most commonly used report toGUS gene and GFP gene. Not used to living but GUS gene, although the GFP genecould live, continuous detection, but the green fluorescence is largely affected bypigment plant itself. In this study, GFP gene can detect in the hair root, only hard to observation in the cotyledons. And novel gene DsRed2report has both inherited theadvantages of GFP gene can live detection, continuous detection, and compared withthe GFP gene, gene DsRed2excitation and light emission wavelength is longer, and itsunder the excitation light reddish, reduce the detection receptor is contained in thechlorophyll pigments such as interference, high signal noise ratio (SNR), within the cellfluorescence is more efficient, easier to test.This study respectively connect GFP gene with DsRed2in plant expression vectorpTF101.1, and contains both carrier and carrier PTF102agrobacterium EHA101infectroot carcinoma3day Jack strains of soybean germination, observation after co-cultureand induction of cotyledon session before the instantaneous positive expression. Theexperimental results show that the GFP gene in cotyledon section parts detected veryhard, but the DsRed2genes can be observed in cotyledon area clear positive bud points,but the positive expression rate is slightly lower than that of GUS gene. Thenrespectively with roots containing the pgfpGUS carrier and carrier pDsRed2GUSagrobacterium K599infect Jack strains of soybean, hair root of positive. Theexperimental results show that the GFP positive can be detected with DsRed2hair root,DsRed2efficiency is slightly lower than that of GUS gene, gene expression with GFPgene.